The inflammatory response governs a wide range of illness from injury to infections and allergies. Initiation of inflammation involves the activation of immune cells that increases synthesis and release of certain cytokines. The released cytokines further activate the appropriate target cells and trigger the phospholipase A2 (PLA2)-involved inflammatory processes. PLA2s are a diverse family of enzymes that hydrolyze the sn-2 fatty acyl bond of phospholipids producing free fatty acids and lysophospholipids (lysoPLs). These enzymes are abundant in pancreatic juice and venoms of snakes and bees. They are also present in small amounts in many types of cells. PLA2s have a wide range of functions involving dietary phospholipid digestion, cellular phospholipid metabolism and turnover, membrane phospholipid remodeling, and the critical roles in the inflammatory processes. PLA2 is a key enzyme regulating the synthesis of a number of bioactive lipid mediators including prostaglandins (PG), thromboxanes (TX) and leukotrienes (LT), collectively called eicosanoids, and platelet activating factor (PAF). Eicosanoids are synthesized from a common precursor of PLA2 enzymatic product, arachidonic acid (AA) released from the sn-2 position of cell membrane phospholipids hydrolyzed by PLA2. These potent lipid mediators play important roles in inflammatory processes and numerous critical illnesses (1, 2). For example, excessive production of these mediators has been linked to inflammation, allergy, brain injury, cancer development and metastasis, and cardiovascular disorders (39, 46, 48). When inflammation occurs in the lung, activated immune response cells release the inflammatory lipid mediators that can induce airway hyperresponsiveness, act as nonspecific chemoattractants to increase leukocyte recruitment, and cause vascular permeability and bronchoconstriction (3, 4). In addition, PLA2 and its enzymatic reaction product lysoPL present in the extracellular fluids may also cause tissue damage (5-7).
Three types of PLA2 have been found in mammalian tissues, the secretory PLA2, the cytosolic PLA2, and the calcium-independent PLA2 (8). At least ten secretory PLA2 isoforms have been identified in the humans and these enzymes have molecular weights around 14 kDa (8-10). Among these proteins the PLA2-IB and PLA2-IIA have been most extensively studied. PLA2-IB is considered as a pancreatic enzyme whose function mainly involves digestion of dietary phospholipids. PLA2-IIA is a non-pancreatic enzyme and has been found to correlate with local and systemic inflammatory responses (11). This enzyme is present in platelets and inflammatory cells including neutrophils and has been found in circulating blood and rheumatoid arthritic synovial fluid (11-13). The primary structure of human PLA2-IIA in platelets and synovial fluid has been determined and its gene cloned (13, 14). Most secretory PLA2 enzymes including PLA2-IB and PLA2-IIA are not specific for AA at the 2-position of phospholipids, however, a recombinant PLA2 of the recently discovered form namely PLA2-X efficiently released AA from adherent mammalian cells (54). Although, the secretory PLA2 enzymes are not specific for AA at the sn-2 position of phospholipids, its enzymatic products of lysoPL and free fatty acid may further activate a cytosolic PLA2 with molecular weight of 85 kDa which specifically releases AA from membrane phospholipids (15, 16). Both PLA2-I and PLA2-II have been implicated in human diseases, particularly the PLA2-IIA in inflammatory diseases (17). High levels of secretory PLA2-IIA have been found in the plasma of patients with acute sepsis, in synovial fluids from patients with arthritis, and in peritoneal fluids from patients with peritonitis (11, 17). PLA2-IIA may also act as an antibacterial agent to destroy bacteria during infection (18). This is because of the nature of the high cationic charge of PLA2-IIA (pI>10.5) that, in conjunction with bactericidal/permeability-increasing protein, PLA2-IIA can readily penetrate the cell wall of gram-negative bacteria and disrupt the anionic bacterial membrane. Expression of pancreatic PLA2 in the lung (19, 20) and its presence, together with PLA2-IIA, in bronchoalveolar lavage fluid (BALF) (21, 22) suggest a role of PLA2-IB in addition to its function as a digestive enzyme. However, the function of the secretory PLA2 in the lung is not clear.
Despite the various isoforms of secretory PLA2, the catalytic reactions of these enzymes, in terms of phospholipid hydrolysis, are the same, i.e., hydrolyzing the fatty acyl group at the sn-2 position of phospholipids at the air/water interface. They require millimolar calcium for their enzymatic reactions and interact strongly with membranes containing anionic phospholipids but interact weakly with an interface composed of zwitterionic phosphatidylcholine (PC) except PLA2-X, which binds tightly to PC vesicles (54). Once PLA2 is secreted into the extracellular fluid, the enzyme has to interact with the outer plasma membrane of cells to exert its action. The interaction between PLA2 and the cell surface may involve the binding of PLA2 with PLA2-specific receptors or with anionic heparan sulfate proteoglycans (HSPG), or by direct interfacial binding and hydrolyzing of membrane phospholipids (23). Interfacial binding is important for plasma membrane fatty acid release catalyzed by secretory PLA2 (24). The major lipid component of the eukaryotic cell outer membrane is PC with a small amount of sphingomylin. Thus, mammalian cells in general are poor substrates for secretory PLA2s. It is not clear how the secretory PLA2s exert their action on cells in terms of phsopholipid hydrolysis without indiscriminately destroying the cells.
Inhibition of lipid mediator production has long been considered for therapeutic purposes (2). However, drugs that have been developed to inhibit production of target lipid mediators or to restrain PLA2 activity have serious side effects and sometimes even exacerbate the pathological conditions. This is, in part, due to the observation that when a target mediator is inhibited, the inhibited pathway often shifts to unwanted over production of another mediator. Also, complexity of the super-family genes of PLA2 makes drug design to control the specific type of PLA2 that is involved in inflammatory disease more difficult (25).
Cystic fibrosis (CF) is caused by the defect of the gene encoding the CF transmembrane conductance regulator (CFTR), a large, membrane-spanning protein that regulates ion flux through the apical surfaces of epithelial cells. Pulmonary complications due to progressive bronchiectasis are the major cause of morbidity and mortality of the CF patients (26). Lung disease in CF is characterized by bacterial infection and intense, neutrophil-dominated inflammation. Lower respiratory tract secretions of most CF patients contain high amounts of proteases, particularly the elastase from polymorphonuclear neutrophils (PMN). The abundant neutrophil elastase (NE) is thought to be a major cause of the epithelial tissue damage that leads to bronchiectasis and bronchial obstruction (27, 28).
The most potent endogenous inhibitor of NE is alpha-1-antitrypsin (α1-AT) (29). The α1-AT in the airspaces is thought to protect fragile bronchoalveolar tissues from destruction by NE. However, intact and functional α1-AT is primarily deficient in the airspaces of patients with CF. α1-AT is a member of the serpin superfamily of proteins. α1-AT is a 52 kDa secreted glycoprotein with 394 amino acid residues that is mainly synthesized by hepatocytes and produced in small amounts by other cells including neutrophils and alveolar macrophages (38). α1-AT is the most abundant proteinase inhibitor in the plasma, and its normal level ranges from 20 μM to 50 μM (38). It inactivates NE in a 1:1 molar ratio to form an α1-AT-NE complex. Inherited α1-AT deficiency is linked to early onset of emphysema and to liver disease (29). In the inflamed CF lung, the deficiency of α1-AT is not due to lack of this protein as occurs in hereditary α1-AT. Rather, the deficiency is due to proteolytic cleavage of the intact α1-AT to yield a truncated 48 kDa α1-AT, which cannot bind and inactivate NE (45). Contrarily, the amount of α1-AT in the serum of most CF patients was more than two-times higher than that in healthy persons, and α1-AT from sera of patients with CF is fully active against NE (50). Although treatment with exogenous α1-AT has been attempted, either by infusion into the systemic circulation or via aerosol inhalation (33), significant clinical benefits of α1-AT replacement have not been demonstrated to date.
It has long been recognized that elevation of AA in the lung of patients with CF is linked to the pathogenesis of chronic lung inflammation (30). High AA is also associated with phospholipids in lung tissue of CFTR gene knockout cftr−/− mice (31), and the high level of AA has been linked to low amounts of phospholipid-bound docosahexaenoic acid (DHA) in involved tissues (32). Epithelial cell lines with the deltaF508 mutation in their CFTR gene also released abnormally high levels of AA when induced by Ca2+ (31). Little is known about the regulation of the production of the high level of AA and the synthesis of the lipid mediators in the CF lung and airway.
The massive influx of neutrophils into infected CF airways is thought to be induced by a number of substances including bacterial products, LTB4 and interleukin (IL)-8 (33). LTB4 is the most abundant eicosanoid found in CF BALF, together with PG and TX. The levels of all of these lipid mediators are markedly elevated in the airways of patients with CF (34). High levels of LTB4 have also been found in sputum and urine of CF children (35) and sputum of CF adults (36). LTB4 not only acts as a chemoattractant to increase leukocyte recruitment, but it also activates neutrophils to release more elastase. LTB4 also induces airway hyperresponsiveness and causes vascular permeability and bronchoconstriction (4). As a result, a cycle of enhanced LTB4 production from AA, chemoattraction of neutrophils, and intense inflammation due to neutrophil flux into lung tissue occurs and further stimulates LTB4 generation from AA, sustaining chronic inflammation and progressively damaging the CF lung. Also, the function of surfactant in the CF lung is impaired, and the surfactant phospholipid level is low. All these seem to suggest that PLA2-mediated inflammation may play a critical role in the CF lung injury.
To investigate whether the increase in AA in bronchial secretions of CF patients is due to the increase in PLA2 activity, Tsao previously discovered that BALF from subjects with CF markedly induced PLA2 activity in vitro (U.S. Pat. No. 6,180,596) (37). This revealed that there might be a PLA2 stimulating factor in the BALFs of CF subjects.